The protective effects of humoral immunity are known to be mediated by a family of structurally related glycoproteins called antibodies. Antibodies initiate their biological activity by binding to antigens. Antibody binding to antigens is generally specific for one antigen and the binding is usually of high affinity. Antibodies are produced by B-lymphocytes. Blood contains many different antibodies, each derived from a clone of B-cells and each having a distinct structure and specificity for antigen. Antibodies are present on the surface of B-lymphocytes, in the plasma, in interstitial fluid of the tissues and in secretory fluids such as saliva and mucous on mucosal surfaces.
All antibodies are similar in their overall structure, accounting for certain similarities in physico-chemical features such as charge and solubility. All antibodies have a common core structure of two identical light chains, each about 24 kilo Daltons, and two identical heavy chains of about 55-70 kilo Daltons each. One light chain is attached to each heavy chain, and the two heavy chains are attached to each other. Both the light and heavy chains contain a series of repeating homologous units, each of about 110 amino acid residues in length which fold independently in a common globular motif, called an immunoglobulin (Ig) domain. The region of an antibody formed by the association of the two heavy chains is hydrophobic. Antibodies, and especially monoclonal antibodies, are known to cleave at the site where the light chain attaches to the heavy chain when they are subjected to adverse physical or chemical conditions. Because antibodies contain numerous cysteine residues, they have many cysteine-cysteine disulfide bonds. All Ig domains contain two layers of beta-pleated sheets with three or four strands of anti-parallel polypeptide chains.
Despite their overall similarity, antibody molecules can be divided into a small number of distinct classes and subclasses based on physicochemical characteristics such as size, charge and solubility, and on their behavior in binding to antigens. In humans, the classes of antibody molecules are: IgA, IgD, IgE, IgG and IgM. Members of each class are said to be of the same isotype. IgA and IgG isotypes are further subdivided into subtypes called IgA1, IgA2 and IgG1, IgG2, IgG3 and IgG4. The heavy chains of all antibodies in an isotype share extensive regions of amino acid sequence identity, but differ from antibodies belonging to other isotypes or subtypes. Heavy chains are designated by the letters of the Greek alphabet corresponding to the overall isotype of the antibody, e.g., IgA contains alpha., IgD contains delta., IgE contains epsilon., IgG contains .gamma., and IgM contains .mu. heavy chains. IgG, IgE and IgD circulate as monomers, whereas secreted forms of IgA and IgM are dimers or pentamers, respectively, stabilized by the J chain. Some IgA molecules exist as monomers or trimers.
There are between 108 and 1010 structurally different antibody molecules in every individual, each with a unique amino acid sequence in their antigen combining sites. Sequence diversity in antibodies is predominantly found in three short stretches within the amino terminal domains of the heavy and light chains called variable (V) regions, to distinguish them from the more conserved constant (C) regions.
Atherosclerosis is a chronic disease that causes a thickening of the innermost layer (the intima) of large and medium-sized arteries. It decreases blood flow and may cause ischemia and tissue destruction in organs supplied by the affected vessel. Atherosclerosis is the major cause of cardiovascular disease including myocardial infarction, stroke and peripheral artery disease. It is the major cause of death in the western world and is predicted to become the leading cause of death in the entire world within two decades.
The disease is initiated by accumulation of lipoproteins, primarily low-density lipoprotein (LDL), in the extracellular matrix of the vessel. These LDL particles aggregate and undergo oxidative modification. Oxidized LDL is toxic and cause vascular injury. Atherosclerosis represents in many respects a response to this injury including inflammation and fibrosis.
In 1989 Palinski and coworkers identified circulating autoantibodies against oxidized LDL in humans. This observation suggested that atherosclerosis may be an autoimmune disease caused by immune reactions against oxidized lipoproteins. At this time several laboratories began searching for associations between antibody titers against oxidized LDL and cardiovascular disease. However, the picture that emerged from these studies was far from clear. Antibodies existed against a large number of different epitopes in oxidized LDL, but the structure of these epitopes was unknown. The term “oxidized LDL antibodies” thus referred to an unknown mixture of different antibodies rather than to one specific antibody. T cell-independent IgM antibodies were more frequent than T-cell dependent IgG antibodies.
Antibodies against oxidized LDL were present in both patients with cardiovascular disease and in healthy controls. Although some early studies reported associations between oxidized LDL antibody titers and cardiovascular disease, others were unable to find such associations. A major weakness of these studies was that the ELISA tests used to determine antibody titers used oxidized LDL particles as ligand. LDL composition is different in different individuals, the degree of oxidative modification is difficult both to control and assess and levels of antibodies against the different epitopes in the oxidized LDL particles can not be determined. To some extent, due to the technical problems it has been difficult to evaluate the role of antibody responses against oxidized LDL using the techniques available so far, but, however, it is not possible to create well defined and reproducable components of a vaccine if one should use intact oxidized LDL particles.
Another way to investigate the possibility that autoimmune reactions against oxidized LDL in the vascular wall play a key role in the development of atherosclerosis is to immunize animals against its own oxidized LDL. The idea behind this approach is that if autoimmune reactions against oxidized LDL are reinforced using classical immunization techniques this would result in increased vascular inflammation and progressive of atherosclerosis. To test this hypothesis rabbits were immunized with homologous oxidized LDL and then induced atherosclerosis by feeding the animals a high-cholesterol diet for 3 months.
However, in contrast to the original hypothesis immunization with oxidized LDL had a protective effect reducing atherosclerosis with about 50%. Similar results were also obtained in a subsequent study in which the high-cholesterol diet was combined with vascular balloon-injury to produce a more aggressive plaque development. In parallel with our studies several other laboratories reported similar observations. Taken together the available data clearly demonstrates that there exist immune reactions that protect against the development of atherosclerosis and that these involves autoimmunity against oxidized LDL.
These observations also suggest the possibility of developing an immune therapy or “vaccine” for treatment of atherosclerosis-based cardiovascular disease in man. One approach to do this would be to immunize an individual with his own LDL after it has been oxidized by exposure to for example copper. However, this approach is complicated by the fact that it is not known which structure in oxidized LDL that is responsible for inducing the protective immunity and if oxidized LDL also may contain epitopes that may give rise to adverse immune reactions.
The identification of epitopes in oxidized LDL is important for several aspects:
First, one or several of these epitopes are likely to be responsible for activating the anti-atherogenic immune response observed in animals immunized with oxidized LDL. Peptides containing these epitopes may therefore represent a possibility for development of an immune therapy or “atherosclerosis vaccine” in man. Further, they can be used for therapeutic treatment of atheroschlerosis developed in man.
Secondly, peptides containing the identified epitopes can be used to develop ELISAs able to detect antibodies against specific structure in oxidized LDL. Such ELISAs would be more precise and reliable than ones presently available using oxidized LDL particles as antigen. It would also allow the analyses of immune responses against different epitopes in oxidized LDL associated with cardiovascular disease.
U.S. Pat. No. 5,972,890 relates to a use of peptides for diagnosing atherosclerosis. The technique presented in said U.S. patent is as a principle a form of radiophysical diagnosis. A peptide sequence is radioactively labelled and is injected into the bloodstream. If this peptide sequence should be identical with sequences present in apolipoprotein B it will bind to the tissue where there are receptors present for apolipoprotein B. In vessels this is above all atherosclerotic plaque. The concentration of radioactivity in the wall of the vessel can then be determined e.g., by means of a gamma camera. The technique is thus a radiophysical diagnostic method based on that radioactively labelled peptide sequences will bound to their normal tissue receptors present in atherosclerotic plaque and are detected using an external radioactivity analysis. It is a direct analysis method to identify atherosclerotic plaque. It requires that the patient be given radioactive compounds.
Published studies (Palinski et al., 1995, and George et al., 1998) have shown that immunisation against oxidised LDL reduces the development of atherosclerosis. This would indicate that immuno reactions against oxidised LDL in general have a protecting effect. The results given herein have, however, surprisingly shown that this is not always the case. E.g., immunisation using a mixture of peptides #10, 45, 154, 199, and 240 gave rise to an increase of the development of atherosclerosis. Immunisation using other peptide sequences, e.g., peptide sequences #1, and 30 to 34 lacks total effect on the development of atherosclerosis. The results are surprising because they provide basis for the fact that immuno reactions against oxidised LDL, can protect against the development, contribute to the development of atherosclerosis, and be without any effect at all depending on which structures in oxidised LDL they are directed to. These findings make it possible to develop immunisation methods, which isolate the activation of protecting immuno reactions. Further, they show that immunisation using intact oxidised LDL could have a detrimental effect if the particles used contain a high level of structures that give rise to atherogenic immuno reactions.
The technique of the present invention is based on quite different principles and methods. In accordance with claim 1 the invention relates to antibodies raised against oxidized fragments of apolipoprotein B, which antibodies are used for immunisation against cardiovascular disease.
As an alternative to active immunisation, using the identified peptides described above, passive immunisation with pre-made antibodies directed to the same peptides is an attractive possibility. Such antibodies may be given desired properties concerning e.g. specificity and crossreactivity, isotype, affinity and plasma halflife. The possibility to develop antibodies with predetermined properties became apparent already with the advent of the monoclonal antibody technology (Milstein and Köhler, 1975 Nature, 256:495-7). This technology used murine hybridoma cells producing large amounts of identical, but murine, antibodies. In fact, a large number of preclinical, and also clinical trials were started using murine monoclonal antibodies for treatment of e.g. cancers. However, due to the fact that the antibodies were of non-human origin the immune system of the patients recognised them as foreign and developed antibodies to them. As a consequence the efficacy and plasma half-lives of the murine antibodies were decreased, and often side effects from allergic reactions, caused by the foreign antibody, prevented successful treatment.
To solve these problems several approaches to reduce the murine component of the specific and potentially therapeutic antibody were taken. The first approach comprised technology to make so called chimearic antibodies where the murine variable domains of the antibody were transferred to human constant regions resulting in an antibody that was mainly human (Neuberger et al. 1985, Nature 314:268-70). A further refinement of this approach was to develop humanised antibodies where the regions of the murine antibody that contacted the antigen, the so called Complementarity Determining Regions (CDRs) were transferred to a human antibody framework. Such antibodies are almost completely human and seldom cause any harmful antibody responses when administered to patients. Several chimearic or humanised antibodies have been registered as therapeutic drugs and are now widely used within various indications (Borrebaeck and Carlsson, 2001, Curr. Opin. Pharmacol. 1:404-408).
Today also completely human antibodies may be produced using recombinant technologies. Typically large libraries comprising billions of different antibodies are used. In contrast to the previous technologies employing chimearisation or humanisation of e.g. murine antibodies this technology does not rely on immunisation of animals to generate the specific antibody. In stead the recombinant libraries comprise a huge number of pre-made antibody variants why it is likely that the library will have at least one antibody specific for any antigen. Thus, using such libraries the problem becomes the one to find the specific binder already existing in the library, and not to generate it through immunisations. In order to find the good binder in a library in an efficient manner, various systems where phenotype i.e. the antibody or antibody fragment is linked to its genotype i.e. the encoding gene have been devised. The most commonly used such system is the so called phage display system where antibody fragments are expressed, displayed, as fusions with phage coat proteins on the surface of filamentous phage particles, while simultaneously carrying the genetic information encoding the displayed molecule (McCafferty et al., 1990, Nature 348:552-554). Phage displaying antibody fragments specific for a particular antigen may be selected through binding to the antigen in question. Isolated phage may then be amplified and the gene encoding the selected antibody variable domains may optionally be transferred to other antibody formats as e.g. full length immunoglobulin and expressed in high amounts using appropriate vectors and host cells well known in the art.
The format of displayed antibody specificities on phage particles may differ. The most commonly used formats are Fab (Griffiths et al., 1994. EMBO J. 13:3245-3260) and single chain (scFv) (Hoogenboom et al., 1992, J Mol Biol. 227:381-388) both comprising the variable antigen binding domains of antibodies. The single chain format is composed of a variable heavy domain (VH) linked to a variable light domain (VL) via a flexible linker (U.S. Pat. No. 4,946,778). Before use as analytical reagents, or therapeutic agents, the displayed antibody specificity is transferred to a soluble format e.g. Fab or scFv and analysed as such. In later steps the antibody fragment identified to have desireable characteristics may be transferred into yet other formats such as full length antibodies.
Recently a novel technology for generation of variability in antibody libraries was presented (WO98/32845, Soderlind et al., 2000, Nature BioTechnol. 18:852-856).
Antibody fragments derived from this library all have the same framework regions and only differ in their CDRs. Since the framework regions are of germline sequence the immunogenicity of antibodies derived from the library, or similar libraies produced using the same technology, are expected to be particularly low (Soderlind et al., 2000, Nature BioTechnol. 18:852-856). This property is expected to be of great value for therapeutic antibodies reducing the risk for the patient to form antibodies to the administered antibody thereby reducing risks for allergic reactions, the occurrence of blocking antibodies, and allowing a long plasma half-life of the antibody. Several antibodies derived from recombinant libraries have now reached into the clinic and are expected to provide therapeutic drugs in the near future.
Thus, when met with the challenge to develop therapeutic antibodies to be used in humans the art teaches away from the earlier hybridoma technology and towards use of modern recombinant library technology (Soderlind et al., 2001, Comb. Chem. & High Throughput Screen. 4:409-416). It was realised that the peptides identified (PCT/SE02/00679), and being a integral part of this invention, could be used as antigens for generation of fully human antibodies with predetermined properties. In contrast to earlier art (U.S. Pat. No. 6,225,070) the antigenic structures i.e. the peptides used in the present invention were identified as being particularly relevant as target sequences for therapeutic antibodies (PCT/SE02/00679). Also, in the present invention the antibodies are derived from antibody libraries omitting the need for immunisation of lipoprotein deficient mice to raise murine antibodies (U.S. Pat. No. 6,225,070). Moreover, the resulting antibodies are fully human and are not expected to generate any undesired immunological reaction when administered into patients.
The peptides used, and previously identified (PCT/SE02/00679) are the following:
TABLE 1A. High IgG, MDA-differenceP 11.FLDTVYGNCSTHFTVKTRKG(SEQ ID NO: 39) P 25.PQCSTHILQWLKRVHANPLL(SEQ ID NO: 40) P 74.VISIPRLQAEARSEILAHWS(SEQ ID NO: 41) B. High IgM, no MDA-differenceP 40.KLVKEALKESQLPTVMDFRK(SEQ ID NO: 42) P 68.LKFVTQAEGAKQTEATMTFK(SEQ ID NO: 43) P 94.DGSLRHKFLDSNIKFSHVEK(SEQ ID NO: 44) P 99.KGTYGLSCQRDPNTGRLNGE(SEQ ID NO: 45) P 100.RLNGESNLRFNSSYLQGTNQ(SEQ ID NO: 46) P 102.SLTSTSDLQSGIIKNTASLK(SEQ ID NO: 47) P 103.TASLKYENYELTLKSDTNGK(SEQ ID NO: 48) P 105.DMTFSKQNALLRSEYQADYE(SEQ ID NO: 49) P 177.MKVKIIRTIDQMQNSELQWP(SEQ ID NO: 50) C. High IgG, no MDA differenceP 143.IALDDAKINFNEKLSQLQTY(SEQ ID NO: 51) P 210.KTTKQSFDLSVKAQYKKNKH(SEQ ID NO: 52) D. NHS/AHP, IgG-ak > 2, MDA-differenceP 1.EEEMLENVSLVCPKDATRFK(SEQ ID NO: 53) P 129.GSTSHHLVSRKSISAALEHK(SEQ ID NO: 54) P 148.IENIDFNKSGSSTASWIQNV(SEQ ID NO: 55) P 162.IREVTQRLNGEIQALELPQK(SEQ ID NO: 56) P 252.EVDVLTKYSQPEDSLIPFFE(SEQ ID NO: 57) E. NHS/AHP, IgM-ak > 2, MDA-differenceP 301.HTFLIYITELLKKLQSTTVM(SEQ ID NO: 58) P 30.LLDIANYLMEQIQDDCTGDE(SEQ ID NO: 59) P 31.CTGDEDYTYKIKRVIGNMGQ(SEQ ID NO: 60) P 32.GNMGQTMEQLTPELKSSILK(SEQ ID NO: 61) P 33.SSILKCVQSTKPSLMIQKAA(SEQ ID NO: 62) P 34.IQKAAIQALRKMEPKDKDQE(SEQ ID NO: 63) P 100.RLNGESNLRFNSSYLQGTNQ(SEQ ID NO: 64) P 107.SLNSHGLELNADILGTDKIN(SEQ ID NO: 65) P 149.WIQNVDTKYQIRIQIQEKLQ(SEQ ID NO: 66) P 169.TYISDWWTLAAKNLTDFAEQ(SEQ ID NO: 67) P 236.EATLQRIYSLWEHSTKNHLQ(SEQ ID NO: 68) F. NHS/AHP, IgG-ak < 0.5, no MDA-differenceP 10.ALLVPPETEEAKQVLFLDTV(SEQ ID NO: 69) P 45.IEIGLEGKGFEPTLEALFGK(SEQ ID NO: 70) P 111.SGASMKLTTNGRFREHNAKF(SEQ ID NO: 71) P 154.NLIGDFEVAEKINAFRAKVH(SEQ ID NO: 72) P 199.GHSVLTAKGMALFGEGKAEF(SEQ ID NO: 73) P 222.FKSSVITLNTNAELFNQSDI(SEQ ID NO: 74) P 240.FPDLGQEVALNANTKNQKIR(SEQ ID NO: 75)or an active site of one or more of these peptides.
In Table 1 above, the following is due:
(A) Fragments that produce high levels of IgG antibodies to MDA-modified peptides (n=3),
(B) Fragments that produce high levels of IgM antibodies, but no difference between native and MDA-modified peptides (n=9),
(C) Fragments that produce high levels of IgG antibodies, but no difference between native and MDA-modified peptides (n=2),
(D) Fragments that produce high levels of IgG antibodies to MDA-modified peptides and at least twice as much antibodies in the NHP-pool as compared to the AHP-pool (n=5),
(E) Fragments that produce high levels of IgM antibodies to MDA-modified peptides and at least twice as much antibodies in the NHP-pool as compared to the AHP-pool (n=11), and
(F) Fragments that produce high levels of IgG antibodies, but no difference between intact and MDA-modified peptides but at least twice as much antibodies in the AHP-pool as compared to the NHP-pool (n=7).